1. Field of the Invention
The present invention relates to the field of biologically-active somatotropin compositions. More particularly, it concerns biologically-active compositions of somatotropin formulated for extended release into the bloodstream of an animal following parenteral administration, methods of preparing these compositions, and methods of using the same.
2. Technical Problem Addressed by the Invention
Although prolonged activity of some biologically active (bioactive) polypeptides can be achieved by parenterally administering only very small doses, others are required in sufficient serum concentrations and/or have such a short half-life in serum that a substantial dose must be administered to provide the desired biological effect over an extended time such as a week or longer. Somatotropins (growth hormones) are an example of such polypeptides.
To prevent undesirably rapid release into an animal""s bloodstream, certain polypeptides have been parenterally administered in liquid vehicles which may optionally contain hydration retardants (antihydration agents) or in association with metals or metal compounds that further lower their solubility in body fluids. To avoid the need for unacceptably large quantities of such a vehicle, and for other reasons including superior prolonged release performance, it is advantageous to employ substantial concentrations of the polypeptide in the vehicle, e.g., as shown in U.S. Pat. No. 5,739,108 to James C. Mitchell, U.S. Pat. No. 4,977,140, assigned to Eli Lilly, U.S. Pat. No. 5,520,927, assigned to Lucky, Ltd., and U.S. Pat. No. 5,744,163, assigned to LG Chemicals Ltd. However, there has been a need to improve the efficiency with which such polypeptides are released into the animal""s bloodstream in a biologically active form (xe2x80x9cbioavailabilityxe2x80x9d) and/or, in some utilities, their effectiveness in providing the desired physiological response in the animal (xe2x80x9cefficacyxe2x80x9d). Each of these factors can substantially affect the amount of the polypeptide that must be administered to achieve the desired biological effect, and consequently, the cost of each administration. Typically, polypeptides such as somatotropins are made in prokaryotic organisms that have been transformed using recombinant DNA, such that even small quantities are very expensive to produce in the pure forms required for product safety and regulatory approval.
3. Description of Related Art
There is currently a substantial body of work which addresses the need for protein formulations which provide for extended release of biologically active polypeptides, including somatotropins. This body of work includes a number of publications describing the use of various stabilizing compounds and excipients. Furthermore, various methods of and devices for administering the bioactive compositions have also been reported in the existing art. Exemplary publications which address this technological problem include the following:
Christensen et al., WO 97/03692, discloses a formulation of growth hormone with zinc, and optionally lysine or calcium, ions. The formulation can contain an excipient such as a disaccharide, a polysaccharide, or a sugar alcohol. Growth hormone so formulated showed resistance to deamidation.
Dong et al., WO 00/13674, discloses a mechanism for timed-release of a drug. The mechanism comprises a semipermeable walled container that houses a capsule, which capsule comprises a drug formulation, a piston, and an osmotic composition. The dosage mechanism releases the drug formulation through a passageway at a controlled rate over a period of up to 24 hours.
Ekwuribe, U.S. Pat. Nos. 5,359,030, 5,438,040, and 5,681,811 disclose a stabilized conjugated peptide complex comprising a peptide conjugatively coupled to a polymer including lipophilic and hydrophilic moieties which is suitable for both parenteral and non-parenteral administration.
Ferguson et al., U.S. Pat. No. 4,977,140, discloses a sustained release formulation comprising bovine somatotropin in a carrier comprising a wax (about 1%-20% by weight) and an oil (about 80%-99% by weight). On injecting into a dairy cow, the formulation led to greater milk production for 28 days.
Hamilton et al., U.S. Pat. No. 4,816,568, discloses compositions of animal growth hormones and stabilizers. The stabilizers are soluble in aqueous solutions, and generally are very polar. The stabilizers taught include polyols, amino acids, amino acid polymers with charged side groups at physiological pH, and choline derivatives. An aqueous formulation of the composition can be formed by (i) dispersing the stabilizer in an aqueous solution and (ii) subsequently adding the growth hormone. A solid formulation can be formed by (i) mixing the stabilizer and the growth hormone, (ii) optionally adding adjuvants, binders, etc. to the composition, and (iii) compressing the composition to form a tablet or pellet.
Kim et al., U.S. Pat. No. 5,520,927, discloses a parenterally administered, slow releasing bioactive pharmaceutical composition comprising somatotropin, at least one tocopherol compound, and a release delaying agent.
Kim et al., U.S. Pat. No. 5,744,163, discloses a formulation for the sustained release of animal growth hormone. The formulation comprises coating somatotropin containing pellets with a film of biodegradable polymer and a poloxamer.
Magruder et al., U.S. Pat. No. 5,034,229, discloses a device for delivering a beneficial agent, e.g. a growth hormone, to an animal. The device can also deliver a polyol as a viscosity modulating means.
Martin, EP 0 216 485, discloses a method of preparing growth hormones complexed with transition metals. Methods for promoting growth in animals by treating them with transition metal complexed growth hormones are also described.
Mitchell, U.S. Pat. No. 5,739,108, discloses extended-release formulations of bioactive polypeptides comprising the polypeptide at from about 10% by weight to about 50% by weight in a dispersion in a biocompatible oil. The polypeptide can be associated with a non-toxic metal or metal salt. The formulation can also comprise an antihydration agent, such as aluminum monostearate.
Pikal, et al., U.S. Pat. No. 5,612,315, discloses formulations for the parenteral administration of human growth hormone comprising human growth hormone, glycine, and mannitol. The disclosed formulations are described as providing stabilization against protein aggregation.
Raman et al., U.S. Pat. No. 5,356,635, discloses a sustained release composition comprising a biologically active agent, e.g. somatotropin; a biodegradable, amorphous carbohydrate glass matrix, throughout which the e.g. somatotropin is dispersed; and a hydrophobic substance. The amorphous carbohydrate glass matrix comprises an amorphous carbohydrate and a recrystallization retarding agent, and makes up from about 60% by weight to 90% by weight of the composition. The composition is solid down to at least about 18xc2x0 C.
Raman et al., WO 93/13792, discloses an implantable device comprising a transition metal-somatotropin complex in combination with a transition metal-solubilizing substance. The transition metal can be zinc, manganese, or copper. The metal-solubilizing substance can be an amino acid. Sucrose can be used to stabilize the somatotropin. The device can comprise silicone tubing or wax.
Seely et al., WO 93/19773, discloses aqueous solutions comprising (i) a lyophilized somatotropin composition comprising somatotropin and arginine HCl and (ii) a diluent comprising EDTA, nonionic surfactant, and optionally buffer or a non-buffering agent such as sucrose or trehalose.
Sivaramakrishnan et al., U.S. Pat. No. 5,219,572, discloses a device for controlled release of macromolecular proteins, e.g. somatotropin. The device comprises a water-soluble outer capsule completely surrounding an inner compartment containing non-uniform beadlets. The beadlets comprise a wax shell which surrounds a core matrix. The core matrix comprises e.g. somatotropin and optionally excipients, stabilizers, binders, and the like, e.g. magnesium stearate or sucrose. Upon dissolution of the outer capsule in the fluid environment in an animal, the beadlets are exposed to the fluid environment, and rupture at various times after exposure.
Sorensen et al., WO 93/12812, teaches that growth hormone can be stabilized by the presence of histidine or a histidine derivative. If the growth hormone is lyophilized, the composition can also comprise a bulking agent, i.e. sugar alcohols, disaccharides, and mixtures thereof.
Sorensen et al., U.S. Pat. No. 5,849,704, discloses a pharmaceutical formulation comprising a growth hormone and histidine or a derivative of histidine as an additive or buffering substance added to provide stability against deamidation, oxidation or cleavage of the peptide bonds in the growth hormone. Also disclosed is that crystallization of growth hormone in the presence of histidine or a derivative thereof gives rise to a higher yield of crystals having higher purity than known methods.
Steber et al., EP 0 523 330 A1, discloses a compacted, indented, partially-coated, implantable composition comprising a biologically active polypeptide (e.g. somatotropin); a fat, wax, or mixture thereof; and a sugar (e.g. mono-, di-, or trisaccharides).
Storrs, et al. U.S. Pat. No. 5,986,073, discloses a method for purifying and recovering biologically active somatotropin monomers. This work is based on the discovery that somatotropin monomers and somatotropin oligomers having overlapping isolelectric points may nevertheless be separated by selective precipitation over a very narrow pH range. Undesirable impurities are removed by this process and the purified somatotropin monomers recovered are suitable for parenteral application to target animals without further purification.
Tyle, U.S. Pat. No. 4,857,506, discloses a multiple water-in-oil-in-water emulsion for the sustained release of a growth hormone. The growth hormone is dispersed in an internal aqueous phase; the internal aqueous phase is dispersed in a water-immiscible liquid or oil phase; and the water-immiscible phase is dispersed in an external aqueous phase. The internal aqueous phase can include up to 40% by weight polyol, glycol, or sugar.
Viswanathan et al., U.S. Pat. No. 4,917,685, discloses a delivery device for a stabilized animal growth hormone. The device comprises a wall which surrounds and defines a reservoir. At least a portion of the wall is porous, to allow passage of growth hormone and stabilizer. The growth hormone and stabilizer formulation is substantially that disclosed by Hamilton et al., described above.
Despite the efforts described in the publications summarized above, there is still room for significant improvement of the technology. The present invention satisfies this need by providing improved, sustained release formulation of a somatotropin which has the advantages of providing both higher and more sustained levels of somatotropin in the serum of animals treated with these formulations.
The art summarized above discloses formulations in which biologically active polypeptides (such as somatotropins) are dispersed in aqueous or non-aqueous vehicles which can be parenterally administered to animals for prolonged release of the polypeptide into the animal""s bloodstream. This invention discloses novel non-aqueous formulations for such parenteral administration, in which a somatotropin (xe2x80x9cSTxe2x80x9d) is dispersed, e.g., suspended, together with at least a first bioavailability enhancing constituent (hereinafter xe2x80x9cBECxe2x80x9d) which enhances the desired biological effect(s) of the ST in the animal. In a specific example, injection of dairy cattle with formulations of this invention have been found to result in unexpectedly elevated levels of serum ST in the animals, and for surprisingly prolonged periods of time, which is normally expected to result in a surprisingly increased production of milk by those cattle. In even more preferred embodiments, the compositions of this invention contain a second BEC, chemically distinguishable from the aforementioned first BEC, which further enhances the desired biological effects of the ST and, in many embodiments, provides with the first BEC a surprisingly great enhancement of those effects. In a particular embodiment of the invention, the first BEC, when used in combination with such a second BEC, prolongs the release enhances the effects of the ST to a highly unexpected extent.
This invention provides compositions of matter which, when administered parenterally to animals, e.g., warm-blooded animals such as mammals, result in higher serum levels of somatotropin (xe2x80x9cSTxe2x80x9d), and in many instances, for periods of time that are longer than those levels and time periods that result from similar administration of previously-available formulations containing the same dose of ST. Accordingly, these compositions of matter provide superior efficacy for inducing weight gain and/or milk production in mammals, compared with previously available formulations containing the same amount of ST. In some embodiments, the compositions of this invention are especially effective for sustaining milk production of a mammal, which has been elevated by administration of a ST, for time periods longer than those provided by the use of previously available ST formulations.
The compositions of the present invention, which include a ST and a first bioavailability-enhancing constituent (hereinafter xe2x80x9cBECxe2x80x9d), enhance the xe2x80x9cbioavailabilityxe2x80x9d of the ST which, as the net result of the release, absorption, elimination, degradation and other physiological phenomena of the ST, typically results in a desired biological effect such as growth, milk production or feed efficiency (e.g., feed-to-milk conversion efficiency). This first BEC typically increases the solubility of the ST in the aqueous body fluids of the animal to be treated, and can be chosen from compounds and substances that are normally regarded as surfactants (preferably non-ionic surfactants, e.g., one or a mixture of two or more of the following: polyoxyethylene fatty acid esters, poloxamers, polyoxyethylene sorbitan fatty acid esters, tocopherol polyethylene glycol succinates and other tocopherol polyalkylene glycol esters of fatty acids containing from about 8 to about 24 carbon atoms, sugar fatty acid esters, polyoxyethylene glycerides, and polyoxyethylene vegetable oils), or from one or a mixture of cyclodextrin compounds such as, for instance, unsubstituted cyclodextrin or cyclodextrin having at least one water solubility-enhancing substituent, e.g., a hydroxyalkyl substituent, or from a mixture of such surfactant(s) and cyclodextrin compound(s). It is not clear whether the characteristics typically regarded as surfactant properties play a predominant, or even important role in the ST bioavailability-enhancing activity of such a first BEC in this invention. In some instances in which the first BEC and ST are combined prior to the ST being lyophilized or spray dried, it appears that the first BEC may function as a protectant of the activity of the ST during such processing and thus enhance the bioavailability of the ST after subsequent parenteral administration.
Optionally, and desirably in many instances, the compositions of this invention include a second BEC as mentioned hereinbefore. This second BEC can be one or a mixture of two or more of (a) an amino acid, amino acid derivative such as histidine-HCl, or an amino acid polymer, such as a polyhistidine; (b) an hydroxamate, such as suberohydrxamic acid, or an hydroxamate derivative, such as a histidine hydroxamate; (c) a non-reducing carbohydrate, such as a polyol or polyol ester; (d) a salt of an oxo-acid, such as a mixture of monobasic and diabasic sodium phosphates; or (e) imidazole or imidazole-HCl.
The present invention also includes methods for making the described compositions of matter and methods of using these compositions (for example, by parenteral administration of the compositions to animals).
In various embodiments of the invention, the ST and the BEC(s) are suspended or otherwise combined in a substantially non-aqueous hydrophobic carrier (oil), yielding a formulation which is fluidly injectable at the body temperature of the animal to be injected (typically 37-39xc2x0 C. for a mammal), and desirably at the ambient temperature for injection of the animal. The compositions of the instant invention may be of any viscosity which is compatible with use of the compositions in accordance with the present invention. In a preferred embodiment, the viscosity of the composition is between about 500 and about 10,000 centipoise at 141s-1. A particularly preferred carrier (vehicle) for use in the present invention is a mixture of 95% sesame oil and 5% aluminum monostearate. Other carriers that are believed to be satisfactorily useful in the invention include alpha-tocopherol acetate and mixtures of vegetable (e.g., sesame or peanut) oils and beeswax.
Unless specifically stated otherwise, percentages of constituents in compositions of the invention described herein are by weight of the composition.
According to the present invention the ST used in the formulations may be from any origin which is suitable for use with the invention, including, but not limited to, native and/or recombinant bovine, porcine, equine, or human somatotropin. The ST used may be present in an essentially pure form or may be combined with another substance (e.g, the ST used may be in the form of a zinc salt or zinc complex of ST).
The compositions of this invention which do not contain a second BEC usually contain a first BEC (or mixture of two or more first BECs) in a concentration of at least about 0.2%, more typically at least about 0.5%, generally not more than about 10%, and more commonly not more than about 5%. The compositions of this invention which do contain a second BEC normally contain a first BEC (or a mixture of two or more first BECs) in a concentration of at least 0.1%, more commonly at least about 0.2%, usually not more than about 10%, and preferably not more than about 5%.
In an alternative embodiment, where the second BEC is an amino acid, amino acid derivative, hydroxamate or hydroxamate derivative, or salt of an oxo-acid, the second BEC comprises at least about 1%, preferably, at least about 2%, by weight of the total composition. Typically, when the second BEC is an amino acid, amino acid derivative it comprises not more than about 15%, preferably, not more than about 10%, by weight, of the total composition.
According to another embodiment of the present invention, when the second BEC used is a non-reducing carbohydrate it comprises at least about 1%, preferably, at least about 3%, by weight of the total composition. Typically, when the second BEC is a non-reducing carbohydrate, it comprises not more than about 20%, preferably, not more than about 15%, by weight, of the total composition.
The present invention also provides a method of preparing the described compositions of matter. According to such an embodiment of the present invention the ST is provided as a lyophilized dry solid (for an example of preparing the lyophilized ST see U.S. Pat. No. 5,013,713, which is incorporated herein by reference), the first BEC is likewise provided as a liquid, in a paste- or wax-like state or a dry solid. In embodiments comprising both a first and a second BEC, either the first BEC, the second BEC or both the first and second BEC can be provided as liquid, in a paste- or wax-like state or a dry solids. The method for preparing these compositions of matter comprises mixing the first BEC, in a liquid state, in a paste- or wax-like state, or a dry solid, with the hydrophobic carrier, to produce a first suspension or solution, as the case may be, and then mixing the lyophilized, dry, solid ST with the first suspension to produce a second suspension. In embodiments having both a first liquid, in a paste- or wax-like state or a dry solid BEC and a second dry, solid BEC, both BECs would be mixed with the hydrophobic carrier to produce the above first suspension.
Another embodiment of the present invention provides a method for inducing improved weight gain or elevated milk production in a mammal. This method comprises injecting an ST formulation of the present invention into the target mammal.
Yet another embodiment of the present invention provides a method for sustaining the elevated milk production response in a lactating mammal. This method comprises injecting the target mammal with a biocompatible ST formulation according to the present invention, wherein the ST present in the formulation is active in the target mammal.
According to the present invention these compositions and methods may be better understood by a review of the detailed description in conjunction with the drawings, which serve to facilitate the further illustration of certain aspects and/or certain embodiments of the invention.
DEFINITIONS
The following definitions are provided in order to aid those skilled in the art to understand the detailed description of the present invention.
Throughout the specification, unless otherwise indicated, percentages of compositions are by weight and temperatures are in degrees Celsius (xc2x0 C.).
As used in the specification and claims, the term xe2x80x9csubstantially non-aqueousxe2x80x9d means essentially anhydrous or containing water in such low proportion that it does not intolerably accelerate release of the polypeptide in the animal. Although this proportion of water may vary with each composition of the invention it is most commonly less than about 2% and most typically less than about 1%.
The term xe2x80x9cnon-toxicxe2x80x9d as used herein refers to components of compositions that are reasonably safe and/or innocuous when used in appropriate amounts and under appropriate conditions in parenteral administration of such compositions as are described herein.
The term xe2x80x9cbiologically-activexe2x80x9d or xe2x80x9cbioactivexe2x80x9d polypeptide or protein (e.g somatotropin) is used herein to describe a polypeptide or protein, which following appropriate parenteral administration to an animal, has a demonstrable effect on a biological process of that animal. The effect may be hormonal, nutritive, therapeutic, prophylactic, or otherwise, and may mimic, complement, or inhibit a naturally occurring biological process. Although there is a vast array of potentially regulatable biological activities or processes, the following are mentioned as exemplary: stimulation of growth, stimulation of lactation, stimulation of egg or offspring production, and enhancement of the efficiency of feed usage.
The term xe2x80x9cbiocompatiblexe2x80x9d, as used herein, refers to substances which have no intolerable adverse effect on the somatotropin, the animal, or, in the case of animals whose products enter the food chain, the consumers of such products.
The term xe2x80x9cPOESxe2x80x9d means polyoxyethylene stearate, the term xe2x80x9cPOE4Sxe2x80x9d means polyoxyethylene 4 stearate, and the term xe2x80x9cPOE8Sxe2x80x9d means polyoxyethylene 8 stearate where the number in the names means the approximate polymer length in oxyethylene units.
xe2x80x9cSustaining elevated milk production responsexe2x80x9d or xe2x80x9csustained elevated milk production responsexe2x80x9d, as used herein, refers to the ability of lactating animals to maintain an elevated level of milk production, over a period of time, despite decreasing serum levels of somatotropin.